On Chip Sorting of Stem Cell-Derived ß Cell Clusters Using Traveling Surface Acoustic Waves.

There is a critical need for sorting complex materials, such as pancreatic islets of Langerhans, exocrine acinar tissues, and embryoid bodies. These materials are cell clusters, which have highly heterogeneous physical properties (such as size, shape, morphology, and deformability). Selecting such materials on the basis of specific properties can improve clinical outcomes and help advance biomedical research. In this work, we focused on sorting one such complex material, human stem cell-derived ß cell clusters (SC-ß cell clusters), by size. For this purpose, we developed a microfluidic device in which an image detection system was coupled to an actuation mechanism based on traveling surface acoustic waves (TSAWs). SC-ß cell clusters of varying size (∼100-500 µm in diameter) were passed through the sorting device. Inside the device, the size of each cluster was estimated from their bright-field images. After size identification, larger clusters, relative to the cutoff size for separation, were selectively actuated using TSAW pulses. As a result of this selective actuation, smaller and larger clusters exited the device from different outlets. At the current sample dilutions, the experimental sorting efficiency ranged between 78% and 90% for a separation cutoff size of 250 µm, yielding sorting throughputs of up to 0.2 SC-ß cell clusters/s using our proof-of-concept design. The biocompatibility of this sorting technique was also established, as no difference in SC-ß cell cluster viability due to TSAW pulse usage was found. We conclude the proof-of-concept sorting work by discussing a few ways to optimize sorting of SC-ß cell clusters for potentially higher sorting efficiency and throughput. This sorting technique can potentially help in achieving a better distribution of islets for clinical islet transplantation (a potential cure for type 1 diabetes). Additionally, the use of this technique for sorting islets can help in characterizing islet biophysical properties by size and selecting suitable islets for improved islet cryopreservation.

Banff 2022 pancreas transplantation multidisciplinary report: Refinement of guidelines for T cell-mediated rejection, antibody-mediated rejection and islet pathology. Assessment of duodenal cuff biopsies and noninvasive diagnostic methods.

The Banff pancreas working schema for diagnosis and grading of rejection is widely used for treatment guidance and risk stratification in centers that perform pancreas allograft biopsies. Since the last update, various studies have provided additional insight regarding the application of the schema and enhanced our understanding of additional clinicopathologic entities. This update aims to clarify terminology and lesion description for T cell-mediated and antibody-mediated allograft rejections, in both active and chronic forms. In addition, morphologic and immunohistochemical tools are described to help distinguish rejection from nonrejection pathologies. For the first time, a clinicopathologic approach to islet pathology in the early and late posttransplant periods is discussed. This update also includes a discussion and recommendations on the utilization of endoscopic duodenal donor cuff biopsies as surrogates for pancreas biopsies in various clinical settings. Finally, an analysis and recommendations on the use of donor-derived cell-free DNA for monitoring pancreas graft recipients are provided. This multidisciplinary effort assesses the current role of pancreas allograft biopsies and offers practical guidelines that can be helpful to pancreas transplant practitioners as well as experienced pathologists and pathologists in training.

Lumped parameter liver simulation to predict acute haemodynamic alterations following partial resections.

Partial liver resections are routinely performed in living donor liver transplantation and to debulk tumours in liver malignancies, but surgical decisions on vessel reconstruction for adequate inflow and outflow are challenging. Pre-operative evaluation is often limited to radiological imaging, which fails to account for post-resection haemodynamic alterations. Substantial evidence suggests post-surgical increase in local volume flow rate enhances shear stress, signalling hepatic regeneration, but excessive shear stress has been postulated to result in small for size syndrome and liver failure. Predicting haemodynamic alterations throughout the liver is particularly challenging due to the dendritic architecture of the vasculature, spanning several orders of magnitude in diameter. Therefore, we developed a mathematical lumped parameter model with realistic heterogeneities capturing inflow/outflow of the human liver to simulate acute perfusion alterations following surgical resection. Our model is parametrized using clinical measurements, relies on a single free parameter and accurately captures established perfusion characteristics. We quantify acute changes in volume flow rate, flow speed and wall shear stress following variable, realistic liver resections and make comparisons with the intact liver. Our numerical model runs in minutes and can be adapted to patient-specific anatomy, providing a novel computational tool aimed at assisting pre- and intra-operative surgical decisions for liver resections.

Nanoparticle-Based Interventions for Liver Transplantation.

Liver transplantation is the only treatment for hepatic insufficiency as a result of acute and chronic liver injuries/pathologies that fail to recover. Unfortunately, there remains an enormous and growing gap between organ supply and demand. Although recipients on the liver transplantation waitlist have significantly higher mortality, livers are often not allocated because they are (i) classified as extended criteria or marginal livers and (ii) subjected to longer cold preservation time (>6 h) with a direct correlation of poor outcomes with longer cold ischemia. Downregulating the recipient's innate immune response to successfully tolerate a graft having longer cold ischemia times or ischemia-reperfusion injury through induction of immune tolerance in the graft and the host would significantly improve organ utilization and post-transplant outcomes. Broadly, technologies proposed for development aim to extend the life of the transplanted liver through post-transplant or recipient conditioning. In this review, we focus on the potential benefits of nanotechnology to provide unique pre-transplant grafting and recipient conditioning of extended criteria donor livers using immune tolerance induction and hyperthermic pre-conditioning.

Pancreatic islet cryopreservation by vitrification achieves high viability, function, recovery and clinical scalability for transplantation.

Pancreatic islet transplantation can cure diabetes but requires accessible, high-quality islets in sufficient quantities. Cryopreservation could solve islet supply chain challenges by enabling quality-controlled banking and pooling of donor islets. Unfortunately, cryopreservation has not succeeded in this objective, as it must simultaneously provide high recovery, viability, function and scalability. Here, we achieve this goal in mouse, porcine, human and human stem cell (SC)-derived beta cell (SC-beta) islets by comprehensive optimization of cryoprotectant agent (CPA) composition, CPA loading and unloading conditions and methods for vitrification and rewarming (VR). Post-VR islet viability, relative to control, was 90.5% for mouse, 92.1% for SC-beta, 87.2% for porcine and 87.4% for human islets, and it remained unchanged for at least 9 months of cryogenic storage. VR islets had normal macroscopic, microscopic, and ultrastructural morphology. Mitochondrial membrane potential and adenosine triphosphate (ATP) levels were slightly reduced, but all other measures of cellular respiration, including oxygen consumption rate (OCR) to produce ATP, were unchanged. VR islets had normal glucose-stimulated insulin secretion (GSIS) function in vitro and in vivo. Porcine and SC-beta islets made insulin in xenotransplant models, and mouse islets tested in a marginal mass syngeneic transplant model cured diabetes in 92% of recipients within 24-48 h after transplant. Excellent glycemic control was seen for 150 days. Finally, our approach processed 2,500 islets with >95% islets recovery at >89% post-thaw viability and can readily be scaled up for higher throughput. These results suggest that cryopreservation can now be used to supply needed islets for improved transplantation outcomes that cure diabetes.

HLA-G1+ Expression in GGTA1KO Pigs Suppresses Human and Monkey Anti-Pig T, B and NK Cell Responses.

The human leukocyte antigen G1 (HLA-G1), a non-classical class I major histocompatibility complex (MHC-I) protein, is a potent immunomodulatory molecule at the maternal/fetal interface and other environments to regulate the cellular immune response. We created GGTA1-/HLAG1+ pigs to explore their use as organ and cell donors that may extend xenograft survival and function in both preclinical nonhuman primate (NHP) models and future clinical trials. In the present study, HLA-G1 was expressed from the porcine ROSA26 locus by homology directed repair (HDR) mediated knock-in (KI) with simultaneous deletion of α-1-3-galactotransferase gene (GGTA1; GTKO) using the clustered regularly interspersed palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) (CRISPR/Cas9) gene-editing system. GTKO/HLAG1+ pigs showing immune inhibitory functions were generated through somatic cell nuclear transfer (SCNT). The presence of HLA-G1 at the ROSA26 locus and the deletion of GGTA1 were confirmed by next generation sequencing (NGS) and Sanger's sequencing. Fibroblasts from piglets, biopsies from transplantable organs, and islets were positive for HLA-G1 expression by confocal microscopy, flow cytometry, or q-PCR. The expression of cell surface HLA-G1 molecule associated with endogenous ß2-microglobulin (ß2m) was confirmed by staining genetically engineered cells with fluorescently labeled recombinant ILT2 protein. Fibroblasts obtained from GTKO/HLAG1+ pigs were shown to modulate the immune response by lowering IFN-γ production by T cells and proliferation of CD4+ and CD8+ T cells, B cells and natural killer (NK) cells, as well as by augmenting phosphorylation of Src homology region 2 domain-containing phosphatase-2 (SHP-2), which plays a central role in immune suppression. Islets isolated from GTKO/HLA-G1+ genetically engineered pigs and transplanted into streptozotocin-diabetic nude mice restored normoglycemia, suggesting that the expression of HLA-G1 did not interfere with their ability to reverse diabetes. The findings presented here suggest that the HLA-G1+ transgene can be stably expressed from the ROSA26 locus of non-fetal maternal tissue at the cell surface. By providing an immunomodulatory signal, expression of HLA-G1+ may extend survival of porcine pancreatic islet and organ xenografts.

Vitrification and Nanowarming of Kidneys.

Vitrification can dramatically increase the storage of viable biomaterials in the cryogenic state for years. Unfortunately, vitrified systems ≥3 mL like large tissues and organs, cannot currently be rewarmed sufficiently rapidly or uniformly by convective approaches to avoid ice crystallization or cracking failures. A new volumetric rewarming technology entitled "nanowarming" addresses this problem by using radiofrequency excited iron oxide nanoparticles to rewarm vitrified systems rapidly and uniformly. Here, for the first time, successful recovery of a rat kidney from the vitrified state using nanowarming, is shown. First, kidneys are perfused via the renal artery with a cryoprotective cocktail (CPA) and silica-coated iron oxide nanoparticles (sIONPs). After cooling at -40 °C min-1 in a controlled rate freezer, microcomputed tomography (µCT) imaging is used to verify the distribution of the sIONPs and the vitrified state of the kidneys. By applying a radiofrequency field to excite the distributed sIONPs, the vitrified kidneys are nanowarmed at a mean rate of 63.7 °C min-1 . Experiments and modeling show the avoidance of both ice crystallization and cracking during these processes. Histology and confocal imaging show that nanowarmed kidneys are dramatically better than convective rewarming controls. This work suggests that kidney nanowarming holds tremendous promise for transplantation.

Origin, transmission, diagnosis and management of coronavirus disease 2019 (COVID-19).

Coronavirus has emerged as a global health threat due to its accelerated geographic spread over the last two decades. This article reviews the current state of knowledge concerning the origin, transmission, diagnosis and management of coronavirus disease 2019 (COVID-19). Historically, it has caused two pandemics: severe acute respiratory syndrome and Middle East respiratory syndrome followed by the present COVID-19 that emerged from China. The virus is believed to be acquired from zoonotic source and spreads through direct and contact transmission. The symptomatic phase manifests with fever, cough and myalgia to severe respiratory failure. The diagnosis is confirmed using reverse transcriptase PCR. Management of COVID-19 is mainly by supportive therapy along with mechanical ventilation in severe cases. Preventive strategies form the major role in reducing the public spread of virus along with successful disease isolation and community containment. Development of a vaccine to eliminate the virus from the host still remains an ongoing challenge.

Perioperative risk factors for pulmonary complications after non-cardiac surgery.

BACKGROUND AND AIMS: Postoperative pulmonary complications (PPCs) lead to increased morbidity, mortality, length of hospital stay, and cost to the patient. This study was conducted to determine the risk factors and assess the incidence of PPC after non-cardiac surgery. MATERIAL AND METHODS: This prospective, observational study was conducted on 1,170 patients undergoing non-cardiac surgery. Details of patient, surgical, and anesthetic factors were collected and patients were followed up for the entire duration of hospital stay for the occurrence of PPC. Assess Respiratory Risk in Surgical Patients in Catalonia (ARISCAT) score and the length of hospital stay was noted for all the patients. Regression analysis was used to find the risk factors associated with development of respiratory complications. RESULTS: The incidence of PPC was found to be 59 in 1,170 patients (5%) in our hospital. Multivariate analysis revealed that patients with intermediate and high risk ARISCAT scoring had higher odds of developing PPC. Higher age (>50 years), positive cough test, presence of nasogastric tube, and intraoperative pulmonary complications were identified as independent risk factors associated with the occurrence of PPC. CONCLUSION: We found 5% incidence of PPC in our study. Recognition of the delineated risk factors and routine use of ARISCAT score for preoperative assessment may help identify patients at a higher risk of developing postoperative pulmonary complications.

Elevated Neutrophil-Lymphocyte Ratio in Luminal-Type Locally Advanced Breast Cancer to Circumvent Neo-Adjuvant Chemotherapy.

Neutrophil-Lymphocyte Ratio (NLR) provides an understanding of the systemic inflammatory conditions. NLR plays an important role as a predictor of mortality in breast and other malignancies. The application of NLR to predict prognosis of Locally Advanced Breast Cancer (LABC) has not been well developed. In this retrospective study, we establish a relationship of pre-treatment NLR with the Pathological Complete Response (pCR) in LABC patients to enhance decision-making and treatment protocols. Data of women diagnosed with carcinoma breast between January 2015 and December 2017 was retrieved from hospital records of a tertiary medical centre in Bangalore, India, after obtaining institutional ethical clearance. LABC patients were categorized into pCR(+) and pCR(-). NLR was calculated and divided into quartiles. The cutoff NLR was determined using the Receiver Operating Characteristic (ROC) curve. Statistical analysis was performed on 119 LABC patients, of which 25 (21%) achieved pCR. Oestrogen Receptor (ER) positivity was significantly lower in pCR(+) than in pCR(-) (p = 0.012). NLR of 2.46 (AUC, 0.744; 95% CI [0.201-0.584]; p = 0.056) was considered the optimum cutoff for pCR(+). A sensitivity of 54%, specificity of 8%, positive predictive value of 1% and high Negative Predictive Value (NPV) of 84% was achieved in the study. A relationship between pCR and the pre-treatment NLR determined a significantly high NPV. Poor pCR in luminal A/B subtype presents with elevated NLR. Therefore, in luminal type A/B (ER- and PR-positive) with elevated NLR (poor outcome) and low pCR (poor response to NACT), the decision of eliminating NACT could be considered, thereby recommending surgical intervention.

Correlation of CD4 counts with the FNAC patterns of tubercular lymphadenitis in patients with HIV: a cross sectional pilot study.

BACKGROUND: Fine needle aspiration cytology (FNAC) of TB lymphadenitis (TBL) in HIV shows four different patterns, which may be reflective of immune status. We hypothesize that the CD(4) counts, a marker of immunologic status, correlates with FNAC morphology of TBL. This study was undertaken to compare the mean CD(4) counts across the different cytology patterns and to correlate the CD(4) counts with FNAC patterns in these patients. METHODS: Forty newly diagnosed HIV patients with TBL on FNAC (10 in each pattern) were selected by convenient sampling based on inclusion exclusion criteria. The CD(4) counts were obtained in these patients. Its correlation with different FNAC patterns was assessed using SPSS version 16. RESULTS: Analysis of covariance showed significant difference in the mean CD(4) counts between all the four patterns [F (374), df (3), P-value = 0.000]. Spearman's correlation analysis showed significant correlation of CD(4) counts with the FNAC patterns (correlation coefficient of 0.967; P-value of 0.01) with pattern 1 having low CD(4) counts and pattern 4 having high CD(4) counts. CONCLUSION: CD(4) counts show significant correlation with FNAC patterns of TBL in HIV patients. Pattern 1, suggestive of poor immunological response (chiefly necrosis, occasional ill defined granuloma, AFB 3+) had low CD(4) counts, while pattern 4, suggestive of good immunological response (well defined granuloma, no necrosis and AFB 1+), had high CD(4) counts. Thus FNAC patterns may be used to predict the CD(4) counts in HIV patients where CD(4) facilities are not available or vice versa.